The constellation of techniques known as recombinant DNA technology includes among its basic elements processes for cutting DNA strands and also for joining DNA strands. By appropriate means, specific fragments of DNA from one organism can be covalently joined with specific fragments derived from a wholly unrelated organism. Such composite, or recombinant, DNA can, under appropriate conditions be transferred to and replicated in a microorganism, thereby conferring upon that organism genetic properties which it would be extremely unlikely to acquire by normal biological mating processes. The potential for practical application of recombinant DNA technology is enormous and far reaching.
DNA is a linear polymer composed of nucleotide subunits. DNA in its native form is made up of two polynucleotide strands of complementary base sequence wound around each other in a right handed double helix, as is well known in the art. DNA may exist in the form of straight chains having two ends or in the form of endless loops. Loops may be converted to straight chains and straight chains may be converted to shorter fragments by the introduction of double-strand chain breaks, which may be produced for example by hydrolysis of the phosphodiester bonds linking adjacent nucleotides. Specific break points, susceptible to one or more enzymes of the type known as restriction endonucleases, may occur in a given nucleotide sequence. Where the strand breaks occur opposite each other on the component strands of the double helix, the break results in blunt-ended strands. However, if the individual strand breaks are staggered by a distance of a few nucleotides, the resulting molecules will have single-stranded, self-complementary ends, sometimes termed cohesive ends.
DNA molecules may be joined end to end by reactions catalyzed by enzymes generically termed ligases. Certain ligases are specific for the joining of DNA molecules having cohesive ends. Others are also capable of joining blunt-ended molecules. In a reaction mixture in which it is desired to join DNA molecules having specific sequences derived from different sources, a reaction mixture is formed containing molecules of the first sequence together with DNA molecules of the second sequence and the appropriate ligase. The DNA ends potentially capable of being joined, termed reactant ends herein, can join in a variety of combinations. In these circumstances, competing joining reactions other than the desired joining can and do occur. Molecules of the first sequence may join with each other, or individual linear molecules may join head-to-tail to form monomer rings, and other higher order joining reactions may compete with the desired reactions. As a result, formation of the desired recombinant molecule may be a relatively improbable event so that only a small fraction of the product molecules are of the desired type.
Prior art attempts to deal with this problem have been indirect and incomplete. In some cases it has been possible to bias the reaction conditions in such a way that the desired product is more favored, or to rely upon physical separation techniques to separate some of the undesired reaction products. In addition, the sophisticated selection techniques of microbial genetics have made it possible to detect certain low frequency recombinants. Where applicable, the latter technique has been extremely valuable. However, such methods are not applicable in all situations and they are tedious to apply.
In recombinant DNA technology, small autonomously replicating DNA molecules in the form of closed loops, termed plasmids, are exploited. The DNA to be recombined with the plasmid may be obtained in a variety of ways, although Federal safety requirements have made the in vitro formation of DNA complementary to isolated mRNA the method of choice. Such DNA is termed cDNA.
Recombinant plasmids are formed by mixing restriction endonuclease-treated plasmid DNA with cDNA containing end groups similarly treated. In order to minimize the chance that segments of cDNA will form combinations with each other, the plasmid DNA is added in molar excess over the cDNA. In prior art procedures this has resulted in the majority of plasmids circularizing without an inserted cDNA fragment. The subsequently transformed cells contained mainly plasmid and not cDNA recombinant plasmids. As a result, the selection process was very tedious and time consuming. The prior art solution to this problem has been to attempt to devise DNA vectors having a restriction endonuclease site in the middle of a suitable marker gene such that the insertion of a recombinant divides the gene thereby causing loss of the function coded by the gene.